Distinguishing fanged frogs (Limnonectes) species (Amphibia: Anura: Dicroglossidae), from Thailand using high resolution melting analysis

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and Tm values were consistent with those predicted, albeit with a slightly different Tm value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species.

taylori was the only species in the L. kuhlii complex found in the north and L. jarujini was inhabited in the south of Thailand.Recently, the occurrence of L. bannaensis and L. taylori was described from the same location, Bo Kluea, Nan Province, in the northern part of Thailand 5 .Morphologically, these two species are extremely close, and it has been difficult to distinguish their tadpoles, juveniles, and adult females 5 .Accurate and rapid species identification is crucial for assessing distributions and population dynamics, so a new approach is needed to better distinguish them.
Recently, several studies reported applying high resolution melting (HRM) in species identification or authentication.0][11] ), and bacteria (e.g., Refs. 12,13).Basically, HRM analysis characterises nucleic acid samples based on their disassociation behaviour and detects sequence differences in PCR-amplified sequences.Samples are discriminated based on their composition, length, and guanine-cytosine (GC) content 14 .An obvious advantage of HRM is that the analysis is performed immediately after the amplification.Thus, post-PCR procedures such as DNA sequencing, which is not a cost-effective method for developing countries, were not required.However, no studies have focused on designing and developing HRM assays to distinguish Limnonectes species from each other.In this study, the HRM was therefore developed to identify Limnonectes species in Thailand, particularly the two sympatric fanged frogs (L.bannaensis and L. taylori).This identification method could contribute to future ecological and systematic studies of the target species.

Nucleotide analysis
Following detailed morphological comparisons and DNA sequence analyses, we here confirm that twelve Limnonectes specimens from Nan province, three from Ratchaburi province, two from Loei Province, and one from Chiang Mai Province were L. bannaensis, L. jarujini, L. isanensis, and L. taylori, respectively.Sequence data of the six Limnonectes species (L.bannaensis, L. isanensis, L. jarujini, L. megastomias, L. taylori, and L. utara) were then further analysed for evaluation of HRM success.Nucleotide variation and GC content are shown in

Simulation HRM (uMELT Quartz)
Before performing an in vitro HRM, the DNA sequences were input into a simulation HRM called uMELT Quartz to determine the shape of the melting curve for each Limnonectes species.The derivative HRM curves (− dF/ dT) and normalised melting curves generated by the programme are shown in Fig. 3A,B.There were differences among the generated melting curves of each species, although the melting plots of L. bannaensis, L. taylori, and L. utara were highly similar to each other.In addition, as can be seen in Fig. 3B, the melting temperatures (T m ) of the tested samples range from 82.0 to 84.5 °C.The predicted T m of L. megastomias (84.5 °C) was the highest among the six species, while that of L. jarujini (82.0 °C) was the lowest.www.nature.com/scientificreports/

In vitro HRM
The HRM assay using the 16S rRNA primer set developed in this study was designed to distinguish the six Limnonectes species found in Thailand.The results of the melting curve and T m obtained were consistent with those predicted from uMELT Quartz (Fig. 4A,B) with a slightly different T m value and a more discriminatory difference in the melting curve.All tested Limnonectes species were easily differentiated from each other by comparing the differences in the melting curve shapes (Fig. 4A).Melting curves were obtained by plotting the negative derivative of the fluorescence intensity with respect to temperature (− dF/dT) versus temperature (T).
The T m value for each species was automatically obtained by identifying the peak of the corresponding melting curve, T m of the tested samples ranged from 82.40 to 83.38 °C.
The HRM assay was then used to confirm the species of 18 Limnonectes samples by comparing them with the reference samples.Similarly, all samples were determined to be the same species based on morphological identification by experts (Table 2).Based on the generating melting curves, from 18 Limnonectes samples, 12 were found to be L. bannaensis, three were L. jarujini, three were L. isanensis, and one was L. taylori (Figs.5A,B), with a confidence interval > 90%, ranging from 90.86 to 99.09.Moreover, in our assay, the Sanger sequencing analysis was carried out in parallel for all the field samples, and the results showed that 100% accuracy was achieved from the HRM analysis.

Discussion
An accurate and rapid identification is essential to enhance knowledge of their ecology and provide the information needed for appropriate conservation planning 15,16 .Identification at the species level using morphological characteristics for certain animal groups, especially amphibians with cryptic species, can be challenging 17 .As mentioned earlier, Limnonectes are potentially important for biodiversity assessments, but there now appear to be many more cryptic species than previously estimated.Especially in widespread complexes, such as the Limnonectes kuhlii complex, Limnonectes displays an extraordinarily high level of morphological similarity 2 .The recent discovery of two Limnonectes species in Thailand indicates the sympatric occurrence of L. taylori and L. bannaensis and highlights the need for a reliable species identification approach other than using morphological characters 5 .The two sympatric species are hardly distinguished from each other based on their morphology.In addition, species identification of tadpoles, juveniles, and adult females of Limnonectes is still a difficult task.
An alternative species identification approach that has been widely used is DNA barcoding (use of short DNA sequences for identification).Over the last four decades, partial fragments of mtDNA have been developed to be used as DNA barcodes for identifying animals at the species level.The 16S rRNA mitochondrial gene region is one of the most used for amphibians (e.g., Refs. 18,19).Although it seems that the advent of recent technologies has made sequencing DNA faster and cheaper, this is not the case in several developing countries.High resolution melting (HRM) analysis has been a promising molecular tool for species identification as it is rapid, simple, costeffective (requires a fluorescent dye and typical PCR reagents), and a sequencing-free method 20 .HRM analyses samples based on their disassociation behaviour and generates melt curves of product DNA fragments 14 .Samples are discriminated based on their composition, length, and guanine-cytosine (GC) content 21 .In 2019, Everman and Wang 9 utilised the HRM of COI identifiers (COI-HRM) to differentiate anurans.Their findings indicated that HRM is a straightforward and efficient method for distinguishing morphologically similar animal species.Remarkably, the method has not been applied to the Limnonectes kuhlii complex, which consists of species with extremely similar morphology.Here, HRM assays using 16S rRNA primers were designed and developed to distinguish six Limnonectes species found in Thailand.www.nature.com/scientificreports/ In our case, the DNA sequencing was carried out in parallel for the field samples, and thus we can perform nucleotide analysis (finding nucleotide variation and GC content, the two important factors that contribute to the success of HRM) and use them in simulation HRM (uMELT Quartz) to evaluate the success potential of the developed HRM analysis.Sequence variations among the tested Limnonectes species were as high as 25.3%, which was a good indicator for success in HRM analysis.Although the least variation between two species was found to be as small as 6.9% (L.jarujini-L.isanensis and L. jarujini-L.taylori), HRM could still be used to differentiate between them.As not only the variation of the sequences could influence DNA duplex stability but also nucleotide composition.The nearest-neighbour (NN) model shows that the stability of DNA depends on the type and orientation of neighbouring base pairs 22 and thus the NN effect has been considered in DNA melting analysis works and simulation HRM tools that are used to predict melting curves for DNA duplexes of interest, including the uMELT Quartz 23 .
5][26] ).This was also the case here, as the uMELT Quartz showed that the melting curves of all tested Limnonectes species were not identical and clearly separated.The results of the in vitro HRM were comparable to those predicted, with a slightly different T m value, and the melting curves were more different among the six species.This is likely due to the narrower temperature increments (0.1 °C) of in vitro HRM analysis.The narrowest temperature increments that can be set in uMELT Quartz are only 0.5 °C23 .Thus, better resolution was achieved with the in vitro HRM assays.The HRM was found to be useful when morphological-based identification is difficult, and therefore the method will also contribute to future ecological and systematic studies on the target species.

Ethics statement
All procedures were conducted in accordance with the current laws in Thailand on experimental animals and were approved by the safety management committee for experiments of the Institutional Ethical Committee of Animal Experimentation of the University of Phayao, Phayao, Thailand (project number 640204004).The study also followed the recommendations in the ARRIVE guidelines.

Samples
Field surveys were conducted in Chiang Mai, Loei, Nan, Sa Kaeo, and Ratchaburi provinces, Thailand, in December 2017.Eighteen individuals of Limnonectes from a hitherto unknown population were collected (Table 3).Tissue samples of the liver from all specimens were preserved in 95% ethanol for further analysis.Specimens were then fixed with 10% formalin for 24 h and subsequently transferred to 70% ethanol.All fixed specimens were deposited at the University of Phayao.Six specimens with 16S rRNA DNA sequences deposited in GenBank were used as reference samples in HRM analysis (Table 3).All samples were examined and identified by experts in the previous study 5 .

Morphological examination, DNA extraction, and sequencing
All specimens were morphologically examined by experts accordingly Suwannapoom et al. 5 Genomic DNA and Sanger sequencing of all 18 specimens (Lim1-Lim18) were then carried out following Suwannapoom et al. 5 Briefly, DNA was extracted from liver tissues using a standard phenol-chloroform extraction protocol of Sambrook et al. 27

Sequence analysis
Two sequence datasets (L and S) with the same number of species but different sequence lengths were constructed for sequence profile analysis.The sequences of the 16S rRNA region of six Limnonectes species (L.bannaensis, L. isanensis, L. jarujini, L. megastomias, L. taylori, and L. utara) generated in previous study 5 were included in the datasets (Table 3).All sequences in an L dataset contained 498 nucleotides, whereas all sequences in an S

Figure 1 .
Figure 1.Distribution of six currently recognised Limnonectes species in Thailand.

Figure 5 .
Figure 5. HRM results of 18 analysed Limnonectes samples (gray color) with the 16S rRNA primers.(A) Normalised curves and (B) Difference curves all five Limnonectes species comparing with L. bannaensis.

Table 1 .
Sequence divergence and GC content in the mitochondrial 16S rRNA gene between the six species within the L. kuhlii complex.L = 498 bp fragment, S = 245 bp fragment.

Table 2 .
Species determination via HRM comparing with Morphological and sequences analyses.
A partial fragment of the mitochondrial 16S rRNA was amplified by polymerase chain reaction (PCR) using the following primers: 16SAR (5′-CGC CTG TTT AYC AAA AAC AT-3′) and 16SBR (5′-CCG GTY TGA ACT CAG ATC AYGT-3′; Kocher et al., 1989).PCR amplifications were performed in a 25 µL reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for 4 min, 35 cycles of denaturing at 94 °C for 40 s, annealing at 55 °C for 16S rRNA for 30 s, extending at 72 °C for 1 min, and a final extension at 72 °C for 10 min.PCR products were sequenced by an ABI 3730xl DNA automated sequencer with both forward and reverse primers.

Table 3 .
Details of Limnonectes specimens used in HRM assay.*Indicates reference samples in the HRM analysis with their sequence accession numbers.